The pharynx is a neuromuscular pump at the anterior end of the alimentary tract. It is made up of 20 muscle cells, 20 neurons,
and 20 other cells. Pharyngeal activity correlates with food intake. The proper feeding rate, as well as the precise timing
of pharyngeal movements, is required for efficient feeding and likely for survival in nature. For most purposes, pharyngeal
behavioral analysis requires no more than a routine stereomicroscope and a pair of eyes, but accuracy can be increased by
video recording followed by off-line analysis in slow motion. Like other C. elegans behaviors, pharyngeal behavior is sensitive to both the immediate environmental conditions as well as to the history of such
conditions.

The movement of bacteria from outside the worm to the intestine is accomplished by two pharyngeal movements: pumps and isthmus
peristalses. Pumps consist of coordinated contraction-relaxation cycles of the radially-oriented muscles of the corpus, anterior
isthmus, and terminal bulb (Figure 1, 2A and Movie 1). Isthmus peristalsis occurs following one out of every 3-5 pumps and is coupled to the preceding pump (Song and Avery, 2012). During isthmus peristalsis, the lumen of the posterior isthmus opens and closes in an anterior-to-posterior wave (Figure 2B and Movie 1). Peristaltic motion transports bacteria trapped in the anterior isthmus to the grinder in the terminal bulb. The grinder
then crushes bacteria so it can be further digested in the intestine (Movie 2). Although isthmus peristalses are coordinated with pumps, it is easiest to study the two motions separately; we dedicate
a separate section to each motion. Use of the electropharyngeogram method as a tool to study pharyngeal physiology (Raizen and Avery, 1994) is discussed in the Wormbook chapter Electrophysiological recordings from the pharynx. At the end of this chapter, we include an appendix (Table 2) with a list of useful promoters for restrictive pharyngeal expression of transgenes. We also include in the appendix references
for two methods of staining the pharynx (Table 3).

Figure 1: Structure of a pharynx.

Figure 2: Time lapse images of feeding motion of an L1 animal under a high power DIC compound microscope. (A) Pumping: the corpus (seen as an opening of lumen of the anterior pharynx, black arrow) and the terminal bulb (red arrow)
contract and relax synchronously. (B) Isthmus peristalsis: The opening of the lumen of the posterior isthmus is marked with
white arrows. During isthmus peristalsis, the posterior isthmus gradually opens and closes in an anterior to the posterior
wave.

Movie 1: A video of a feeding L1 in the presence of 20 mM serotonin. Isthmus peristalsis occurs every 3-5 pumps.

Movie 2: A video of an adult pumping seen under a dissecting microscope. A grinder motion during pumping in the head is visible
when it is moving back and forth.

1. Pumping

1.1. Measuring pumping by counting grinder movements using a stereomicroscope

During feeding, worms suck in and trap bacteria in the corpus, and grind bacteria in the terminal bulb with a structure called
the grinder (Figure 1). One complete cycle of synchronous contraction and relaxation of the corpus and the terminal bulb is called a pump (Figure 2A, Movie 1). A simple way to measure feeding is to count how many times worms pump in a minute (pumps per minute: ppm). Because grinder
movement in the terminal bulb is easier to see than corpus movement, and because contraction/relaxation cycles are synchronized
along the whole pharynx, pumping rate can be measured by counting grinder movements. A stereomicroscope used for daily maintenance
of worms (examples: Wild M5A, Leica MS5, and Zeiss Stemi 2000) can be used to count pumps at a magnification of 40-50X (Movie 2). Higher magnifications are required for counting pumps of larvae. Observation of pumping in first larval stage worms (L1s)
requires > 100X magnification. Because pumping rate can vary from minute-to-minute or even second-to-second (Hobson et al., 2006), it is sometimes desirable to measure ppm of the same worm several times. This is particularly useful when the worm studied
is a precious one (for example, an animal on which a laser-ablation operation had been performed); in such a case, pumping
rate can be counted for 10-20 seconds every minute for 10 minutes and the median or mean is then used. Another instance where
multiple measurements of the same worm are informative is when a perturbation is predicted to affect the variance rather than
the absolute rate of pumping (Hobson et al., 2006). If the number of worms is not limiting and the key parameter of interest is the mean pumping rate, then we recommend measuring
the behavior of at least 10 worms. Although it is possible to measure pumping rate of a worm among a population of other worms
on the same plate, we find that measurements are more reliable and show reduced variance when worms are individually plated.
It is important to use age-matched worms for comparisons. Feeding is easiest to visualize in 6-24 hour old adults.

Under certain conditions, the pumping rate is too fast to count in real time or the motions of individual pumps are abnormal
and are therefore not clearly discerned. Under these conditions, it is useful to record movies and replay them at one half
to one third of the original speed to count pumping. When using a digital camera, the frame rate should be at least 10 frames
per second. Under rare conditions when pumping rates greater than 300 per minute (5 Hz) are expected, the digitization rate
should be faster, and at least twice the rate of the pumps.

Direct counting using a stereomicroscope can be used to measure pumping rates of the worms under various experimental conditions.
Precise ppm counting of worms cultivated on a full lawn of bacteria that is a low quality food source (e.g. OP50 and its derivative
DA837) is tricky because of increased locomotive activity of hungry worms. In such cases, we recommend plating a single worm
on a bacterial lawn of approximately 5 mm diameter and recording a video. Table 1 shows ppm as a function of food quality or after surgical manipulations.

Table 1: Pumping rates (average ± SEM) as a function of diet and after laser operations.

Neuron type killed

No food

DA837 bacteria

HB101 bacteria

None

41 ± 16

265 ± 20

191 ± 12

MC

10 ± 10

45 ± 27

Not done

1.2. Measuring pumping with high power differential interference contrast (DIC) optics of a compound microscope

Although a simple stereomicroscope is adequate for counting pumps based on grinder movements, a compound microscope equipped
with DIC optics is necessary for observing movements in the corpus and isthmus or for measuring the speed of contraction or
relaxation. Isthmus peristalsis movements can be directly visualized only with high power optics (see below).

Worms are placed on a 2% agar pad with abundant bacteria and 3-5 µl of M9 or NGM buffer. A cover slip is placed on top of
the worms. After a 5-15 minute acclimation period, the worms will begin feeding. They can be viewed with a 40X, 63X, or 100X
objective lens equipped with DIC optics. For prolonged viewing of the same worm, it is necessary to move the stage to keep
the worm (or worm's pharynx) in the field of view. This is difficult to do in adult worms because their movements are too
large to easily track by hand. There are a couple of tricks to minimize movements of large worms while observing feeding under
high power magnification. The first is to use worms with a mutation that impairs locomotion. We commonly use worms with mutations
in the body muscle levamisole receptor subunit unc-29 (Avery, 1993). Severe paralysis of body muscle, as seen with unc-54 mutants, is best to avoid since it appears to interfere with normal feeding behavior (David Raizen, unpublished observations).

A second recently described trick for monitoring feeding behavior while minimizing locomotion is to surround the worm with
a suspension of 0.05-0.1 µm polystyrene beads (Polyscience Corp, Warrington PA) on a high concentration (10%) agar pad beneath
a cover slip (Fang-Yen et al., 2012). The increased friction of the beads provides a high barrier to worm body motions and essentially immobilizes the worm.
However, the pharynx, which is internal to the worm, continues to behave even when the worm is immobile. A 10-15 minute acclimation
period on the pad and in the presence of the beads is required before worms resume pumping, which is stimulated by the addition
of serotonin (5 mg/mL) to the pad. When interpreting experiments in which polystyrene beads are used for immobilization, it
is important to be aware that mechanical input is known to inhibit pumping (Keane and Avery, 2003).

Under low light conditions, grayscale values of neighboring pixels can be binned to increase camera sensitivity. This will
reduce the exposure times of individual frames and allow for faster frame rates. In special cases, for example when measuring
the speed of pharyngeal relaxation in subparts of the pharynx, a camera that is specialized for high speed recording (for
example, Andor iXon camera) capable of frame rates on the order of 1000 frames per second is required (Fang-Yen et al., 2009).

2. Isthmus peristalsis

2.1. Direct observation of isthmus peristalsis using high power DIC optics of a compound microscope

Using a compound microscope, one can measure the frequency and duration of isthmus peristalsis as well as the relative timing
of isthmus peristalses to pumps (Figure 2B, Movie 2). A drawback of this method is that the amount of food given to each worm on the agarose pad cannot be easily controlled
and, since food density affects the frequency of isthmus peristalses (Bo-mi Song, unpublished observation), isthmus peristalsis
rates on agarose pads may be variable. In contrast to food, it is possible to tightly control the concentration of drugs on
the agarose pad. Agarose suspensions are prepared by adding 2% (weight/volume) agarose to an M9 solution containing the drug
at the desired final concentration. Avoid using dimethyl sulfoxide (DMSO) as an organic solvent, since at a concentration
as low as 0.25% DMSO is an activator of isthmus peristalsis (Bo-mi Song, unpublished observations). Ethanol at concentrations
up to 0.25% does not affect either pharyngeal pumping rate or isthmus peristalsis rate, and is therefore a reasonable vehicle
for the drugs. Effects of drugs on isthmus peristalsis rate can be examined by placing worms on the drug-containing pads.
Isthmus peristalsis is best measured in L1s that are at least three hours old because the pharyngeal muscles of younger worms
often twitch. The following is a specific example of the analysis of the effect of a drug (serotonin) on isthmus peristalses.
Other drugs can be studied in a similar fashion.

Protocol: Observation of the effect of serotonin on isthmus peristalsis in 3-5 hour old L1 larvae in the absence of food.

After bleach treatment, transfer embryos suspended in 200 µL of M9 onto an unseeded NGM plate and incubate at room temperature
for 2 hours. Absorption of the M9 into the agar during the 2-hour incubation will result in unhatched embryos sticking to
NGM agar surface, thus facilitating the selective transfer of newly hatched L1s. Transfer L1s within 2 hours of hatching to
obtain worms with a defined age.

Transfer the L1s to an unseeded NGM plate and remove unhatched embryos, if any, by aspiration. Incubate the L1s for 3 hours
at room temperature until they are 3-5 hour old.

Make an agarose pad on a glass slide using approximately 100 µl of the agarose/serotonin solution. Place 1-3 µl of M9 solution
on the pad, transfer 5-10 worms into the solution and put a coverslip on top. After 15 minutes of incubation on the pad, focus
attention on the posterior isthmus and count the number of peristaltic movements.

2.2. Observations of isthmus peristalsis with the aid of fluorescent bacteria or beads using a stereomicroscope

Indirect detection of isthmus peristalsis using fluorescent bacteria or fluorescent latex beads permits the assessment of
the effect of food density on the frequency of isthmus peristalsis on an NGM agar surface. During isthmus peristalsis, bacteria
and other particles in the corpus pass through the lumen of the posterior isthmus. Bacteria expressing fluorescent proteins
can be detected as they pass through the posterior isthmus during a peristalsis (Song and Avery, 2012). When fluorescent latex beads are used for the assay, the ratio between beads and food is important. If the bead concentration
is too low, the fluorescent signal will be too low to reliably identify every isthmus peristalsis, whereas if the bead concentration
is too high, background fluorescence will obscure the signal from the beads.

Blue light increases isthmus peristalsis rate. Thus, bacteria or beads that emit red fluorescence are preferable since the
light used to illuminate red beads is green. We have successfully used 0.5 µm red fluorescent latex beads from Sigma-Aldrich
(L3280). The density of food and the age of worms affect isthmus peristalsis rates. We therefore compare isthmus peristalses
of equal-age worms. To ensure constant bacterial concentration, we plate equal volumes of bacterial suspension of a defined
concentration (defined by optical density at 600 nm) on each plate and incubate for a defined period at the same temperature.
To obtain age-matched 1-day-old adults, we transfer L4 animals the previous day.

Remove bacteria from an individual worm by letting it crawl ~1 minute on an unseeded NGM plate. Transfer the worm to the test
plate seeded with mCherry-expressing HB101 and observe isthmus peristalsis under a stereomicroscope with a rhodamine fluorescence
filter by looking for red signal passing through the posterior isthmus.

3. Measuring food intake using GFP bacteria and BODIPY dye

Feeding can be indirectly measured using bacteria expressing a fluorescent protein. We commonly use GFP-expressing bacteria.
Because worms defecate every 45-50 seconds in the presence of plentiful food (Thomas, 1990), ingested contents stay in the gut only for 5-15 minutes (Leon Avery & Young-Jai You, unpublished data). Therefore, the
intensity of fluorescence in the intestinal lumen depends primarily on the intake of food 5-15 minutes prior to observation.
It follows that one must be careful when studying feeding by this method under experimental conditions that may affect defecation.
An alternative to using GFP bacteria is to use the fluorescent dye BODIPY (You et al., 2008), which has the advantage of assessing feeding over longer time periods (Figure 3). However, BODIPY accumulation can be affected by pumping rate and not only by food ingestion (Young-Jai You, unpublished
observations) and therefore should be used with caution when comparing between intake of foods of different qualities or between
strains with different efficiencies of food transport.

Figure 3: BODIPY staining after 15 minutes of feeding with HB101. (A) Worm was fasted for 12 hours and refed for 15 minutes with HB101 in the presence of BODIPY (B) worm was not fasted but
fed with HB101 in the presence of BODIBY for 15 minutes. Reproduced with permission from (You et al., 2008).

Protocol: Using GFP bacteria or BODIPY to measure food intake

Inoculate GFP-expressing bacteria into a 1:1 mixture of LB and M9 to make a bacterial broth and grow overnight at 37 °C without
shaking. When kept at 4 °C, the broth can be used for approximately one month.

Maintain both wild type and test strains at 20 °C by picking three adults every day to a new plate seeded with HB101.

Place age-matched individual worms on individual plates seeded with a confluent lawn of GFP-expressing bacteria and allow
them to feed ad libitum for the desired period of time. Place a 20 µl drop of 1 M NaN3 on top of each worm to kill it. Treating worms with NaN3 prevents additional feeding while you are transferring the worm to the 2% agar pad and add a cover slip.

Take an image of a whole worm using a GFP filter and a 10X objective lens.

For BODIPY staining, dissolve the BODIPY dye in DMSO at 1 mg/ml to make a stock solution. Dilute the stock solution and spread
on plates to achieve a nominal final concentration of 200 ng/ml (the final concentration of DMSO is 0.02%, which is 10-fold
lower than the concentration that can affect isthmus peristalses). After the desired period of feeding, take pictures and
analyze the fluorescence intensity using Image J.

Caution: Avoid transferring excessive GFP bacteria, since it will result in high background fluorescence and spoil your analysis.

Note that although NaN3 has been reported to bleach GFP fluorescence expressed in C. elegans cells, it has not been a problem for this method.

4. Cessation of feeding: Lethargus

Under typical laboratory conditions, worms rarely stop feeding for more than a few seconds unless in lethargus. Lethargus
is the ~2 hour period before each of the four molts (Singh and Sulston, 1978). Both pumping and isthmus peristalses cease during lethargus. Cessation of feeding therefore specifically identifies worms
in lethargus (Raizen et al., 2008; Van Buskirk and Sternberg, 2007). We use absence of pumping for 10 seconds in the appropriately aged animal to signify lethargus. L3 and L4 lethargus can
be readily identified based on absence of pumping under high magnification (40-50X) of a dissecting microscope. To identify
L1 and L2 lethargus, higher magnification is needed. We typically use >100X magnification of a stereomicroscope. Unlike the
cessation of pumping under conditions of satiety, which is extremely sensitive to environmental conditions (see below), the
cessation of pumping during lethargus is a robust behavior that is insensitive to environmental conditions. We have found
that even when animals are mechanically stimulated to move continuously for 30 minutes during L4 lethargus, the duration of
pumping cessation remains constant at approximately two hours (Robert Driver and David Raizen, unpublished observations).
Any pumping observed during lethargus is therefore abnormal and requires non-behavioral anatomical criteria to define the
stage as lethargus. For an example of inappropriate pumping during lethargus, see Van Buskirk and Sternberg (Van Buskirk and Sternberg, 2007).

Protocol: Identifying worms within 10 minutes of the start of L4 lethargus:

At the start of the day, transfer 30-50 mid to late L4 worms to a plate fully seeded with bacteria.

Every 10 minutes, observe the plates under 10-20X magnification to search for worms that are not moving. If a worm is not
moving, check under 40-50X magnification whether or not it is pumping. If it does not pump for 10 seconds or more, then it
has entered L4 lethargus.

Perform experiments on the worms in lethargus. Note that worms that are not pumping always show morphological evidence of
the molt under DIC optics including a cap at the anterior end of the buccal cavity.

5. Cessation of feeding: Satiety

When worms feed after a period of starvation or when they eat highly-preferred bacteria, they can become quiescent. We call
this “satiety quiescence” because it mimics the behavioral sequence of satiety in other animals and because the behavior depends
on food quality, nutritional status and the history of feeding. The following detailed protocols are modified from a prior
publication (You et al., 2008).

5.1. Preparation of worms and test plates

Worms: Maintain both wild type and test strains by picking three adults every day to a new plate seeded with HB101 at 20 °C.

Caution: Contamination with other bacteria can affect feeding and satiety quiescence. We add Streptomycin (200 µg/ml) to plates
to prevent growth of other E. coli. Also, since worm growth and satiety quiescence is adversely affected on dry plates (Young-Jai You, unpublished observation),
we use plates that are no older than 2 weeks and not dried.

Test plates: Prepare test plates with bacteria from bacterial broth prepared as described above (see protocol: Using GFP bacteria
or BODIPY to measure food intake). One day before the assay, seed 35 mm NGM plates with 5 µl of HB101 bacteria broth in the
center. Ten plates per test strain are needed. The bacteria lawn needs to be small in order to rapidly find the worms while
minimally disturbing them.

Incubate the seeded plates at 37 °C overnight.

After removing the plates from 37 °C, leave them at room temperature for a day before use.

Satiety quiescence can be observed under two conditions: (1) after fasting and subsequent refeeding, and (2) when fed good
quality food. The fasting-refeeding condition shows most consistent satiety quiescence.

5.2. Inducing satiety quiescence using a fasting and refeeding assay

Day 1:

Synchronization of worms: Transfer 30 L4 hermaphrodites from a non-crowded culture plate (At 20 °C, a plate typically becomes
crowded 2-2.5 days after placement of 3 adult wild-type worms on the plate) to a new HB101-seeded plate. L1 synchronization
using bleaching doesn't work for obtaining staged worms for satiety quiescence assay for two reasons. First, L1 bleaching
and synchronization requires overnight starvation after egg prep. This affects the worms’ metabolism (Young-Jai You, unpublished
observation) so it could affect feeding behavior. Second, many mutants grow slower and less synchronized than wild type worms.

Fasting: within 12 hours after transfer, L4 worms become young adults. Transfer young adults individually to 60 mm unseeded
NGM plates (i.e., one worm/plate). To minimize the amount of bacteria, transfer worms that are off the bacteria lawn using
a minimum amount of bacteria on the worm pick. Tapping the plate enhances worm movement thus increasing the chance that they
leave the food. Fast the individually-plated worms at 20 °C for 12 hours.

Day 2:

After 12 hours of fasting, transfer the fasted worms individually to the test plates. Fasted animals should appear pale and
bloated with embryos. Transfer 10 worms for each test group. To minimize the difference in refeeding period among test samples,
we recommend testing no more than 3 groups, 30 worms in total, at one time.

Place the plates next to the microscope. Do not stack the plates and leave enough space between them to allow you to pick
one without touching the others.

Refeed the worms undisturbed for 3 or 6 hours. During this time, minimize vibration and noise around the testing area.

After 3 hours of refeeding, set your microscope magnification to 40 or 50X. Transfer a plate carefully to the microscope base
and observe the worm.

The worm should be quiescent, neither moving nor pumping. Start timing to measure the quiescence duration. The quiescence
duration is defined as the interval between the beginning of observation of the quiescent animal to the time at which the
animal resumes feeding and locomotion.

The first movement a quiescent animal makes is typically a backwards movement, which is followed by resumption of pharyngeal
pumping. Occasionally, a worm will make a sub-second body jerk or pump once or twice but then return to being quiescent. Stop
the timer only when the worm shows sustained movement and pumping for > 3 seconds.

After measuring the quiescence duration, place the plate back next to the microscope while analyzing the next worm. After
an additional 3 hours of refeeding (i.e., 6 hours of refeeding total), measure quiescence duration again.

The phenotype should be scored blind to genotype or other conditions that you are testing.

Caution: quiescence behavior is fragile and sensitive to environmental disturbance. Although we don't know what disturbs worms
while we watch them, light, heat and movement of the plates are all candidates.

5.3. Inducing satiety quiescence using high-quality bacteria

Maintain worms and prepare test plates as described above. Pick 10 L4s per test group and place each on a test plate seeded
with HB101 or Comamonas bacteria. After 18 hours, measure quiescence duration as described above. The phenotype again should be scored blind.

6. Appendix: Pharyngeal markers

Table 2: Restrictive promoters for driving transgene expression in the pharynx. We list promoters that may be useful for selective expression in the pharynx. Promoters that are expressed in only a single
pharyngeal cell type are in bold type.

7. Acknowledgements

We thank Leon Avery for comments. D.M.R is supported by R01 NS064030-01A1 and by NARSAD, B-M S. is supported by HL46154 from
the US public Health Service and Y-J. Y. is supported by 09SDG2150070 from the American Heart Association.